As an energy source for germination to leaf production until rice is capable of carrying out photosynthesis, large quantities of protein, starch, and lipid are accumulated in the albumen tissue of rice seeds. These accumulated materials are very stable, and germination is possible even after a few years of storage at ordinary temperature. Accumulated findings on gene expression regulatory mechanisms and advancement in genetic recombination techniques have lead to many efforts in recent years on the accumulation of useful substances in the seeds of plants (see Non-patent Documents 1 to 3).
Genetic recombination techniques may also be used to produce useful substances in microorganisms or animals, but the use of plants has many advantages (see Non-patent Documents 1 and 2). For example, since plants produce energy by using sun light and carbon dioxide in the atmosphere, they do not consume fossil fuels or pollute the atmosphere, and are benign to the earth's resources and environment. Furthermore, plants represented by rice are highly safe because they do not contain harmful substances to humans such as prions, viruses typical of animals, and toxins of microorganisms in the first place. Furthermore, the cost when using plants is estimated to be only one-tenth to one-fiftieth compared to when using microorganisms or animals. Thus, use of plants is very advantageous.
Because of such advantages, development of second-generation genetically modified crops that contain useful substances with health-related functions is promoted in recent years by using genetic recombination techniques. Known representative examples include cedar pollen allergy-relieving rice in which part of the cedar pollen antigen recognition site (epitope) is expressed in the rice albumen (see Non-patent Documents 4 and 5), and golden rice in which β-carotene is highly accumulated by expressing enzymes derived from maize and bacteria in the rice albumen (see Non-patent Documents 6 and 7).
As described above, plants are recognized for their potential as bioreactors; thus, development of third-generation genetically modified crops that accumulate raw materials of pharmaceuticals such as antibodies or vaccines is expected. Producing useful substances less expensively and in large amounts is very important in advancing the actual realization of third-generation genetically modified crops.
There are multiple factors that determine the amount of exogenous gene products, and the most important factor is expression promoter. This is because the timing, site, and/or amount of exogenous gene expression are regulated by the expression promoter. When rice is used as a host, the exogenous gene products are known to accumulate in larger amount in the albumen rather than in the assimilatory tissues such as leaves and stems (see Non-patent Document 8). Furthermore, it is known that in some cases expression of exogenous genes in leaves, stems, and such has harmful effects on growth. Accordingly, promoters that cause high expression of exogenous genes specifically in the albumen are considered to be useful, and many promoters that induce albumen-specific expression have been isolated and used.
So far, for example, the GluA-2 promoter and GluB-1 promoter have been commonly used as rice-seed-specific expression-inducing promoters (see Non-patent Documents 4 and 6). However, these promoters induce expression mainly in small regions in the outermost layers of the albumen called aleuron layer and subaleurone layer (see Non-patent Document 9).
On the other hand, the Glb-1 promoter, 20 kDa globulin, and 16 kDa allergen promoter are known as promoters that induce expression in internal endosperm; however, because they induce expression in places other than the seed, and induce expression during the vegetative growth stage in addition to the stage of seed formation, they could not be used for induction of exogenous genes that cause growth inhibition. Furthermore, there are problems such as weak promoter activity and their use has been limited (see Non-patent Documents 10 and 11).
Prior art documents relating to the present invention are shown below.
[Prior Art Documents]
[Patent Documents]
    [Patent Document 1] Japanese Patent Application Kokai Publication No. (JP-A) 2004-321079 (unexamined, published Japanese patent application)    [Patent Document 2] JP-A (Kokai) 2002-209462    [Patent Document 3] JP-A (Kokai) 2002-058492    [Patent Document 4] Japanese Patent Kohyo Publication No. (JP-A) 2006-521107 (unexamined Japanese national phase publication corresponding to a non-Japanese international publication)    [Patent Document 5] JP-A (Kohyo) 2006-512067    [Patent Document 6] JP-A (Kohyo) 2004-528022    [Patent Document 7] JP-A (Kohyo) 2003-503033    [Patent Document 8] JP-A (Kohyo) 2002-539824    [Patent Document 9] JP-A (Kohyo) 2001-518305    [Patent Document 10] JP-A (Kohyo) H10-513364    [Patent Document 11] Japanese Patent Saikohyo Publication No. (JP-A) 2004/056993 (unexamined Japanese national phase publication corresponding to a Japanese international publication)    [Patent Document 12] Japanese Patent No. 3149951    [Patent Document 13] Japanese Patent No. 3030339    [Patent Document 14] JP-A (Saikohyo) 01/064865    [Patent Document 15] JP-A (Kohyo) H11-510056    [Patent Document 16] JP-A (Saikohyo) 96/030509    [Patent Document 17] JP-A (Kohyo) H06-506584    [Patent Document 18] JP-A (Kokai) 2002-291484    [Patent Document 19] JP-A (Kokai) 2002-253262    [Patent Document 20] JP-A (Kohyo) 2004-500885    [Patent Document 21] JP-A (Kohyo) 2004-508803    [Patent Document 22] JP-A (Kohyo) 2003-523172    [Patent Document 23] JP-A (Saikohyo) 00/058454    [Patent Document 24] JP-A (Kohyo) 2002-521072    [Patent Document 25] JP-A (Kohyo) 2002-504336    [Patent Document 26] JP-A (Kohyo) 2001-512318    [Patent Document 27] JP-A (Kokai) 2005-168500    [Patent Document 28] JP-A (Kokai) 2005-027654    [Patent Document 29] JP-A (Kokai) 2004-105030    [Patent Document 30] JP-A (Kokai) 2001-292777    [Patent Document 31] JP-A (Kokai) 2001-169790    [Patent Document 32] JP-A (Kohyo) 2003-510040    [Patent Document 33] JP-A (Kokai) 2000-041688    [Patent Document 34] JP-A (Kohyo) 2002-509696    [Patent Document 35] JP-A (Kohyo) 2001-517434    [Patent Document 36] JP-A (Kohyo) 2001-519659    [Patent Document 37] JP-A (Kokai) H10-248570    [Patent Document 38] JP-A (Kohyo) 2001-512322    [Patent Document 39] JP-A (Kohyo) 2000-507108    [Patent Document 40] JP-A (Kohyo) H10-504969[Non-Patent Documents]    [Non-patent Document 1] Daniell, H. et al., “Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants”, Trends Plant Sci, (2001), Vol. 6, p. 219-226.    [Non-patent Document 2] Fischer, R. et al., “Plant-based production of biopharmaceuticals”, Curr Opin Plant Biol, (2004), Vol. 7, p. 152-158.    [Non-patent Document 3] Hartmann, R. and Meisel, H., “Food-derived peptides with biological activity: from research to food applications”, Curr Opin Biotechnol, (2007), Vol. 18, p. 163-169.    [Non-patent Document 4] Takagi, H. et al., “A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses”, Proc Natl Acad Sci USA, (2005a), Vol. 102, p. 17525-17530.    [Non-patent Document 5] Takagi, H. et al., “Oral immunotherapy against a pollen allergy using a seed-based peptide vaccine”, Plant Biotechnol J, (2005b), Vol. 3, p. 521-533.    [Non-patent Document 6] Paine, J. et al., “Improving the nutritional value of Golden Rice through increased pro-vitamin A content”, Nat Biotechnol, (2005), Vol. 23, p. 482-487.    [Non-patent Document 7] Ye, X. et al., “Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm”, Science, (2000), Vol. 287, p. 303-305.    [Non-patent Document 8] Takaiwa, F. et al., “Endosperm tissue is good production platform for artificial recombinant proteins in transgenic rice”, Plant Biotechnol J, (2007), Vol. 5, p. 84-92.    [Non-patent Document 9] Qu, I. Q. and Takaiwa, F., “Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice”, Plant Biotechnol J, (2004), Vol. 2, p. 113-125.    [Non-patent Document 10] Storozhenko, S. et al., “Folate fortification of rice by metabolic engineering”, Nat Biotechnol, (2007), Vol. 25, p. 1277-1279.    [Non-patent Document 11] Wu, C. Y. et al., “Promoters of rice seed storage, protein genes direct endosperm-specific gene expression in transgenic rice”, Plant and Cell Physiology, (1998), Vol. 39, p. 885-889.